U.S. patent application number 10/312047 was filed with the patent office on 2004-06-17 for use of c4-c10 acids for preventing gram-negative bacterial infections.
Invention is credited to Popoff, Michel Yvan.
Application Number | 20040116523 10/312047 |
Document ID | / |
Family ID | 26212484 |
Filed Date | 2004-06-17 |
United States Patent
Application |
20040116523 |
Kind Code |
A1 |
Popoff, Michel Yvan |
June 17, 2004 |
Use of c4-c10 acids for preventing gram-negative bacterial
infections
Abstract
The invention relates to the use of C.sub.4-C.sub.10 acids
and/or of at least one of their salts or esters, for preparing a
pharmaceutical composition intended to prevent Gram-negative
bacterial infections, in particular Salmonella infections.
Inventors: |
Popoff, Michel Yvan;
(Plaisir, FR) |
Correspondence
Address: |
MUSERLIAN AND LUCAS AND MERCANTI, LLP
600 THIRD AVENUE
NEW YORK
NY
10016
US
|
Family ID: |
26212484 |
Appl. No.: |
10/312047 |
Filed: |
February 7, 2003 |
PCT Filed: |
June 22, 2001 |
PCT NO: |
PCT/FR01/01971 |
Current U.S.
Class: |
514/558 ;
514/559 |
Current CPC
Class: |
A61K 31/19 20130101;
Y02A 50/30 20180101; A61K 31/20 20130101; A61P 31/04 20180101; Y02A
50/483 20180101; Y02A 50/481 20180101; A61K 31/192 20130101 |
Class at
Publication: |
514/558 ;
514/559 |
International
Class: |
A61K 031/20 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 22, 2000 |
FR |
00/07992 |
Jun 29, 2000 |
FR |
00/08383 |
Claims
1. Use of an effective amount of at least one compound of formula
(I) as follows: R.sub.1-COO-R.sub.2 (I)in which: R.sub.1 represents
a C.sub.4-C.sub.9 saturated carbon-based chain, optionally
substituted with one or more hydroxyl or amine functions, or with
an aromatic ring; R.sub.2 represents a hydrogen atom, a monovalent
alkali metal atom or an alkyl radical, it being understood that,
when R.sub.2 represents a hydrogen atom and R1 represents C.sub.7
saturated carbon-based chain substituted with an amine function,
then said amine function is not at position 2 or 8; as an active
principle, for preparing a pharmaceutical composition with a
neutral pH intended to prevent Gram-negative bacterial infections,
both in humans and in animals.
2. Use according to claim 1, characterized in that said
Gram-negative bacterial infections are Salmonella infections.
3. Use according to claim 1 or 2, characterized in that the
compounds of formula (I) are chosen from valeric acid, caproic
acid, oenanthic acid, caprylic acid and pelargonic acid, their
monohydroxylated derivatives, and also their salts and their
esters.
4. Use according to any one of the preceding claims, characterized
in that the compounds of formula (I) are chosen from those in which
the R.sub.1 radical represents a carbon-based chain substituted
with a hydroxyl function, said hydroxyl function being at position
2 when the R.sub.1 radical contains 4 to 6 carbon atoms and at
position 2 or 8 when the R.sub.1 radical contains 7 to 9 carbon
atoms.
5. Use according to any one of the preceding claims, characterized
in that the compounds of formula (I) are chosen from caprylic acid,
its derivatives hydroxylated at position 2 or 8, their salts and
their esters.
6. Use according to any one of the preceding claims, characterized
in that the alkali metal atoms defined for the R.sub.2 radical are
chosen from sodium and potassium.
7. Use according to any one of the preceding claims, characterized
in that the R.sub.2 radical is a C.sub.1-C.sub.4 alkyl radical.
8. Use according to claim 6, characterized in that the
C.sub.1-C.sub.4 alkyl radical defined for the R.sub.2 radical is
chosen from methyl, ethyl and butyl radicals.
9. Use of an effective amount of sodium caprylate, as an active
principle, for preparing a pharmaceutical composition with a
neutral pH intended to prevent Gram- negative bacterial infections,
in particular Salmonella infections, in humans or in animals.
10. Use according to any one of the preceding claims, characterized
in that the effective amount of said compounds of formula (I)
corresponds to single doses of between 20 mM and 200 mM.
11. Use according to any one of the preceding claims, characterized
in that the pharmaceutical composition is administered orally.
12. Use according to any one of the preceding claims, characterized
in that the pharmaceutical composition is added to the drinking
water and/or to the feed distributed to farm-stock animals.
Description
[0001] The present invention relates to the use of C.sub.4-C.sub.10
acids for preventing Gram-negative bacterial infections, in
particular Salmonella infections, both in animals and in
humans.
[0002] The Salmonellae are enteroinvasive bacteria which are
pathogenic for humans and animals. The crucial step in the
triggering of salmonellosis, whether it involves gastroenteritis or
a generalized infection of typhoid fever type, is the entry of the
Salmonellae into the ileal epithelial cells.
[0003] It has now been thoroughly demonstrated that the majority of
the genes required for this entry (invasion genes) are grouped
together on the SPI-1 pathogenicity island at centisome 63 on the
Salmonella chromosome (J. E. Galan, Mol. Microbiol., 1996, 20,
263-271). These genes encode the IagA (or HilA) transcriptional
activator, belonging to the OmpR/ToxR family, for the components of
the inv-spa-prg type III secretion apparatus and for its targets
including the SipBCDA (or SspBCDA), StpA (or SptP) and AvrA
proteins. The expression of the iagA gene is, itself, very tightly
controlled.
[0004] The transcription of iagA requires the production of two
derepressors, SprA (or HilC or SirC) and HilD, which are encoded by
SPI-1 genes and form part of the AraC/XylS family (Eichelberg et
al., 1999, Mol. Microbiol., 1999, 33, 139-152; Schechter et al.,
Mol. Microbiol., 1999, 32, 629-642). The transcription of iagA is
also controlled directly or indirectly by two-component systems,
RcsB-RcsC and PhoP-PhoQ, which are not encoded by genes located at
centisome 63. The RcsB-RcsC system responds to osmolarity (Arricau
et al., Mol. Microbiol., 1998, 29, 835-850) and the PhoP-PhoQ
system responds to the concentration of divalent cations, such as
calcium and magnesium ions (Garcia Vescoci et al., Cell, 1996, 84,
165-174; Miller et al., Proc. Natl. Acad. Sci. USA, 1989, 86,
5054-5058) of the environmental medium.
[0005] Other environmental conditions (low oxygen tension, slightly
alkaline pH) are also required for the expression of iagA; however,
the mechanisms involved have not been identified.
[0006] When the osmolarity and the concentration of calcium or
magnesium ions are low (in water for example), the PhoP-PhoQ and
RcsB-RcsC systems repress directly or indirectly the expression of
iagA. In the absence of the IagA activator, the genes encoding the
components and targets of the Inv-Spa-Prg secretion apparatus are
not expressed, and the Salmonellae are incapable of entering into
epithelial cells in culture.
[0007] On the other hand, when the osmolarity and the concentration
of calcium or magnesium ions are high (in the intestinal lumen for
example), the PhoP-PhoQ and RcsB-RcsC systems no longer repress the
expression of iagA.
[0008] It is presumed that the SprA and HilD derepressors, by
binding to the iagA promoter or by interfering with the PhoP or
RcsB regulators, are involved in this regulation mechanism. The
IagA protein will then activate the transcription of the inv, spa
and prg operons. The product of invf, the first gene of the inv
operon, is, itself, a regulator of the AraC family and will
activate the transcription of the sip operon (Kaniga et al., Mol.
Microbiol., 1994, 13, 555-568). At this stage, the components of
the Inv-Spa-Prg secretion apparatus are produced. Its targets, in
particular the Sip proteins, are also synthesized, but they remain
stored in the cytoplasmic compartment since the secretion apparatus
is inactive. The activity of the secretion apparatus will be
triggered by contact with the epithelial cell (Galan, 1996). The
Sip proteins will be secreted and will form a translocator, which
will be used to inject the effector proteins, including StpA and
AvrA, into the cytosol of the target cell.
[0009] These effector proteins then activate various signalling
pathways, causing a variety of cellular responses and, finally, the
entry or the Salmonellae into the cell. However, it has been shown
that these two conditions (osmclarity and high concentration of
divalent cations) must be simultaneously optimal in order for the
Salmonellae to fully express their invasive capacity.
[0010] If just one of these conditions is modified, the invasive
capcapacity of the Salmonellae is then very greatly decreased
(Balaj et al., Mol. Miczobiol.. 1996, 22, 703-714). For this
reason, the Salmonellae cultured in a medium with a low osmolarity
or with a low concentration of divalent cations are incapable of
penetrating into epithelial cells in culture.
[0011] The set of data reported above shows that the expression of
the entry phenotype in Salmonella is very finely controlled by
regulators acting in cascade (probably not all are identified) as a
function of the environmental conditions.
[0012] If just one of these regulators is interfered with, then a
noninvasive phenotype must result therefrom.
[0013] While it is very easy to vary the osmolarity or
concentration of divalent cations of a cell culture medium in
vitro, for the purpose of blocking the invasion of Salmonellae into
these epithelial cells in culture, it is not, however, possible to
carry out the same approach in vivo.
[0014] During the last fifteen years, the number of Salmonella
infections has considerably increased. Each year throughout the
world typhoid fever is responsible for 600 000 deaths for
approximately 20 million cases.
[0015] In France, salmonellosis caused by S. enteritidis represents
approximately 33% of cases of toxic food poisoning.
[0016] The Salmonellae therefore still constitute a major public
health problem. Epidemiological studies have clearly shown that
this upsurge in salmonelloses (except in the case of typhoid fever,
which is a strictly human disease) is due to the consumption of
products of animal origin which are contaminated by Salmonella. One
of the most demonstrative examples is that of S. enteritidis, which
has been the cause of a worldwide epidemic. The source of
contamination by S. enteritidis has been completely identified. It
is eggs and egg-based products.
[0017] The situation risks becoming even more preoccupying with the
generalization of the use of antibiotics in animal feed. The
consequence of this farming practice is the appearance of bacterial
strains which are multiresistant to antibiotics, to such an extent
that the panoply of the various antibiotics available may very
rapidly become insufficient.
[0018] This phenomenon has, moreover, led to drastic measures being
taken in certain cases. For example, for poultry farms, the
European directive 92/117 imposes on Member States a standardized
monitoring of the presence of Salmonella in breeding flocks. Point
V of Annex III specifies that flocks which are recognized to be
contaminated by S. typhimurium or S. enteritidis must be
slaughtered. This provision has recently been modified by directive
97/22, which makes it possible to continue to exploit flocks
contaminated by S. typhimurium or S. enteritidis if it is
established, to the satisfaction of the competent authority, that
the infection due to these two bacteria has disappeared.
[0019] Many investigations have been related to the study of the
bacteriostatic or bactericidal activity of various acylated
monoglycerides and fatty acids.
[0020] Thus, it has been shown (Petschow et al., J. Med. Microbiol.
1998, 47, 383-389) that lauric acid for example, which is a
saturated fatty acid in which the chain is formed by 12 carbon
atoms, exhibits bactericidal activity in vitro on S. typhi, Vibrio
cholerae and Shigella sonnei at the concentration of 0.25 g/l.
[0021] According to this same article, it appears that, at the
concentration of 1.25 g/l, caprylic acid
(CH.sub.3(CH.sub.2).sub.6COOH), which is a short-chain (8 carbon
atoms) saturated fatty acid, has no bactericidal activity in vitro
on S. typhi, Vibrio cholerae, enteropathogenic and enterotoxigenic
E. coli and Shigella sonnei.
[0022] It has also been shown, in vitro, that, at the concentration
of 7.8 mM, caprylic acid has no bacteriostatic or bactericidal
effect on Gram-positive or Gram-negative bacteria, whereas other
fatty acids with a longer, in particular from 12 carbon atoms
upwards, saturated carbon-based chain, such as lauric acid, or some
unsaturated fatty acids, such as for example linoleic acid and
oleic acid, are very effective (Kabara et al., Antimicrob. Agents
Chemother., 1972, 2, 23-31).
[0023] Finally, many authors have demonstrated that certain fatty
acids, such as for example n-hexanoic acid, caprylic acid and
decanoic acid, have, at acid PH, bactericidal activity on a great
variety of Gram-positive or Gram-negative microorganisms and make
it possible to treat various types of infection (US patents 4 489
097 and 4 406 884, patent application EP 0 021 504). Those
documents specify, however, that, in order to be effective, these
acids must be used at acid pH so as to be in free acid form.
[0024] It appears, therefore, that the bacteriostatic or
bactericidal activity of fatty acids is linked to the length of the
carbon-based chain and to the number of unsaturations that it
carries, the most significant activity being attributed to fatty
acids comprising at least 12 carbon atoms, and preferably one or
two unsaturations.
[0025] Faced with this major problem, namely Gram-negative
bacterial infections and in particular salmonelloses, and knowing
that the use of antibiotics in animal feed will very certainly be
prohibited in a few years' time, at least in the Member States of
Europe, there is a real need to develop compositions which make it
possible to prevent Gram-negative bacterial infections, and in
particular Salmonella infections, in order to avoid the use of
antibiotics and the phenomena of resistance.
[0026] It is in order to remedy this problem that, surprisingly,
the inventors have developed what forms the subject of the
invention.
[0027] A subject of the present invention is therefore the use of
an effective amount of at least one compound of formula (I) as
follows:
R.sub.1-COO-R.sub.2 (I)
[0028] in which:
[0029] R.sub.1 represents a C.sub.4-C.sub.9 saturated carbon-based
chain, optionally substituted with one or more hydroxyl or amine
functions, or with an aromatic ring;
[0030] R.sub.2 represents a hydrogen atom, a monovalent alkali
metal atom or an alkyl radical, it being understood that, when
R.sub.2 represents a hydrogen atom and R.sub.1 represents a C.sub.7
saturated carbon-based chain substituted with an amine function,
then said amine function is not at position 2 or 8; as an active
principal, for preparing a pharmaceutical composition with a
neutral pH intended to prevent Gram-negative bacterial infections,
and in particular Salmonella infections, both in humans and in
animals.
[0031] Depending on what the R.sub.2 radical means, the compounds
of formula (I) above can, therefore, be present in the form of an
acid, of a salt or of an ester.
[0032] The inventors have, in particular, demonstrated that the
preventive administration of a composition with a neutral pH
containing at least sodium caprylate to mice subsequently infected
with three major serotypes of Salmonella makes it possible to
considerably decrease the level of splenic colonization by these
bacteria, even though this composition has no bacterial activity at
such a pH.
[0033] According to the invention, the pH of the pharmaceutical
composition used is preferably between 6.5 and 7.5, and even more
preferably between 7.1 and 7.4.
[0034] A pH value which is particularly suitable for the use in
accordance with the invention is one between 7.2 and 7.3.
[0035] Among the compounds of formula (I) above, mention may be
made, in particular, of valeric acid, caproic acid, oenanthic acid,
caprylic acid and pelargonic acid, and their monohydroxylated
derivatives, and also their salts and their esters.
[0036] According to a particular embodiment of the invention, and
when the R.sub.1 radical of the compounds of formula (I) represents
a carbon-based chain substituted with a hydroxyl function, then
said hydroxyl function is at position 2 when the R.sub.1 radical
contains 4 to 6 carbon atoms and at position 2 or 8 when the
R.sub.1 radical contains 7 to 9 carbon atoms. Among these
particular compounds of formula (I), mention may in particular be
made of 2-hydroxycaprylic acid and 8-hydroxycaprylic acid.
[0037] Among the compounds of formula (I), caprylic acid (which
corresponds to a compound of formula (I) in which R.sub.1
represents a C.sub.7 saturated carbon-based chain), its derivatives
hydroxylated at position 2 or 8, their salts and their esters are
particularly preferred.
[0038] In fact, besides its antifungal properties (Wyss et al.,
Arch. Biochem., 1945, 7, 418), caprylic acid used at a neutral pH
makes it possible, unexpectedly, to prevent Salmonella infections,
even though, at such a pH value, this acid has no bactericidal
activity. According to the invention, the alkali metal atoms
defined for the R.sub.2 radical are preferably chosen from sodium
and potassium.
[0039] According to the invention, the alkyl radicals defined for
the R.sub.2 radical are preferably chosen from C.sub.1-C.sub.4
alkyl radicals, among which methyl, ethyl and butyl radicals are
most particularly preferred.
[0040] According to an advantageous embodiment of the invention,
use is made of sodium caprylate, i.e. a compound of formula (I) in
which R.sub.1 represents a C.sub.7 saturated carbon-based chain and
R.sub.2 represents a sodium atom.
[0041] The effective amount of compound(s) of formula (I)
corresponds preferably to single doses of between 20 mM and 200 mM,
and more preferably of between 50 mM and 100 mM.
[0042] The pharmaceutical composition used in accordance with the
invention can also contain one or more additional active
principles.
[0043] The compounds of formula (I) used in accordance with the
invention can, of course, be formulated in a pharmaceutically
acceptable vehicle consisting of one or more excipients
conventionally used for preparing pharmaceutical compositions, such
as anti-aggregating agents, antioxidants, colorants, vitamins,
mineral salts, flavour enhancers, or smoothing, assembling or
isolating agents, and, in general, any excipient conventionally
used in the pharmaceutical industry.
[0044] Of course, those skilled in the art will make sure, in this
instance, that the additive(s) optionally used is (are) compatible
with the intrinsic properties attached to the present invention; in
particular the use of these additives should have no incidence on
the pH of the pharmaceutical compositions used.
[0045] The pharmaceutical composition used according to the present
invention is preferably administered orally, and can be in various
forms, such as in the form of tablets, gelatin capsules, drinkable
suspensions or lozenges, or in any other form suitable for the oral
administration method.
[0046] The pharmaceutical composition used according to the present
invention can, in particular, be added to the drinking water and/or
to the feed distributed to farm-stock animals (poultry, cattle,
pigs, sheep, etc.) so as to decrease the incidence of Gram-negative
bacterial infections, and in particular Salmonella infections, and
limit carrying, and thus reduce the risk of a subsequent
contamination of humans.
[0047] The pharmaceutical composition used according to the present
invention can also be administered to humans as a preventive
medicinal product in order to reduce the risk of infection in
individuals staying for limited periods in a region highly endemic
in particular for salmonelloses.
[0048] The pharmaceutical composition used according to the present
invention, coupled with mass vaccination, and while awaiting the
setting up of immune protection, can also be used in humans to stop
or slow down an epidemic of typhoid fever.
[0049] Besides the preceding arrangements, the invention also
comprises other arrangements which will emerge from the following
description, which refers to an example of demonstration of the
activity of sodium caprylate in preventing salmonelloses in mice,
and also to the attached figures, in which:
[0050] FIG. 1 shows the effect of the concentration of Ca.sup.2+
ions on the expression of the iagA'-lacZ, invF'-lacZ and sipB'-lacZ
fusions;
[0051] FIG. 2 shows the effect of the concentration of sodium
benzoate on the expression of the iagA'-lacZ, invF'-lacZ and
sipB'-lacZ fusions;
[0052] FIG. 3 shows the effect of the concentration of sodium
caprylate on the expression of the iagA'-lacZ, invF'-lacZ and
sipB'-lacZ fusions;
[0053] FIG. 4 shows the effect of sodium benzoate and of sodium
caprylate on the expression of the tviB'-lacZ fusion;
[0054] FIG. 5 shows the effect of a mixture of sodium benzoate and
of sodium caprylate on the splenic colonization of mice infected
orally with S. typhimurium C52.
[0055] It should, however, be clearly understood that this example
is given only by way of illustration of the subject of the
invention, of which it in no way constitutes a limitation.
EXAMPLE 1 : DEMONSTRATION OF THE PREVENTIVE ACTIVITY OF SODIUM
CAPRYLATE ON SALMONELLA INFECTIONS
[0056] I) Materials and methods
[0057] I-a) Strains, media and culture conditions
[0058] The list of Salmonella strains used in this example are
shown in Table I hereinafter:
1 TABLE I Strain Characteristics Origin or reference S. typhi Ty2
Parental strain WHO collection (PS) Reference Felix 59 S. typhi
Ty266 Ty2 carrying the Virlogeux et al., tviB'-lacZcat
Microbiology, 1995, fusion 141, 3039-3047 S. typhi Ty267 Ty2
carrying the Arricau et al., iagA'-lacZcat Molecular fusion
Microbiology, 1998, 29, 835-850 S. typhi Ty272 Ty2 carrying the
Arricau et al., invF'-lacZcat 1998, mentioned fusion above S. typhi
Ty277 Ty2 carrying the Arricau et al., sipB'-lacZcat 1998,
mentioned fusion above S. typhimurium PS, virulent for WHO
collection C52 mice Reference C52 S. dublin 5917 PS, virulent for
WHO collection mice Reference 5917 S. enteritidis PS, virulent for
Thorns et al., LA5 mice Microbial Pathogenesis, 1996 20,
235-246
[0059] The antigenic formula of each Salmonella strain was
controlled by slide agglutination with antisera specific for
somatic and flagellar antigenic factors, sold by the company
BioRad-Diagnostics Pasteur.
[0060] Routinely, the strains were cultured at 37.degree. C. on
agar or in typto-casein-soya broth (TCS; sold by the company
BioRad-Diagnostics Pasteur).
[0061] In order to study the expression of the invasion genes in
vitro, two media which are derived from standard LB medium
(Sambrook et al., Molecular Cloning, 1989) were prepared:
[0062] a medium favourable to the expression of the Salmonella
invasion genes (favourable LB medium), and
[0063] an LB medium unfavourable to the expression of the
Salmonella invasion genes (unfavourable LB medium).
[0064] The standard LB medium of origin contains 170 rnM of NaCl
and 850 .+-.50 .mu.M of Ca.sup.2+ ion.
[0065] The favourable LB medium (high osmolarity and concentration
of Ca.sup.2+ions) was defined as being the standard LB medium
modified so as to have a final NaCl concentration of 300 mM and a
final Ca concentration of 5 mM.
[0066] The unfavourable LB medium (high osmolarity and low
concentration of Ca.sup.2+ ions) was defined as being the standard
LB medium modified so as to have a final NaCl concentration of 300
mM, and is supplemented with 1 mM of ethylene glycol
bis(.beta.-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) so
as to create a divalent cation depletion of the medium.
[0067] The mobility of each strain was studied using the U-tube
culturing technique (Popoff and Le Minor, Guide pour la preparation
des serum anti-Salmonella (Guide for preparing salmonella
antisera], 1997). When necessary, the culture media were
supplemented with chloramphenicol (Cm) at 15 .mu.g/ml.
[0068] I-b) Determination of the .mu.-galactosidase activity
[0069] Each of the strains described above was cultured at
37.degree. C. on agar or in TCS broth. The P-galactosidase activity
was determined using 4-methyl-umbelliferyl-p-D-galactopyranoside
sold by the company Sigma, according to the technique of Klarsfeld
et al. (Mol. Mibrobiol., 1994, 13, 585-597). The results, expressed
in P-galactosidase units (fluorescence per hour and per
OD.sub.600), represent the mean of the values obtained in at least
three independent experiments. The standard deviations were less
than 10% of the values presented.
[0070] I-c) Use of "Biotype 1001".RTM. plates
[0071] A "Biotype 100".RTM. plate sold by the company BioMerieux is
composed of 100 microcupules. Each micro-cupule contains a
dehydrated carbon-based substrate, with the exception of the first
cupule, which is a control without substrate (the amount of
dehydrated substrate per cupule is not given by the
manufacturer).
[0072] Normally, the "Biotype 100".RTM. plate makes it possible to
study the nutritional capacity of a bacterial strain on 99
different sources of carbon (auxanogram).
[0073] In the context of the present example, these plates were
used to search for carbon-based compounds which would block the
expression of the invasion genes in Salmonella.
[0074] For this, the S. typhi strain Ty267, which is a derivative
of the parental strain Ty2 carrying the iagA'-lacZcat
transcriptional fusion (Table I), was cultured in TCS broth for 18
hours at 37.degree. C. with shaking (200 rpm).
[0075] This culture was then diluted 100-fold in favourable LB
medium (0.6 ml of culture for 60 ml of favourable LB), and then
this suspension was used immediately to seed the 100 cupules of a
"Biotype 100".RTM. plate, in a proportion of 450 .mu.l of
suspension per cupule.
[0076] After incubation for 36 hours in an incubator at 37.degree.
C., the activity of the iagA'-lacZcat fusion in the Ty267 strain
was determined for the culture of each cupule.
[0077] By comparing the value of the P-galactosidase activity
measured in a cupule containing a carbon-based substrate with that
of the control cupule without substrate, it was possible to
investigate whether a substrate had no effect on, activated or
repressed the expression of the iagA'-lacZcat fusion.
[0078] I-d) Experimental infection of mice
[0079] The 5-6-week-old female C57Bl/6 mice used in the context of
this experiment originated from the IFFA-CREDO breeding centre
(LArbresle, France).
[0080] Throughout the duration of the experiments, the mice had
access ad libidum to the drinking water which had a given
composition for each experiment.
[0081] They were infected orally in the following way. The
Salmonella strains were cultured in TCS broth for 18 hours at
37.degree. C. with shaking (200 rpm). This culture was then
adjusted to 108 bacteria/ml (in the stationary growth phase, 1
OD.sub.600 =1.5 .times.109 bacteria/ml), and then the number of
viable bacteria was controlled by counting on TCS agar.
[0082] On the day of infection (DO), the drinking-water bottles of
the mice were replaced with bottles containing 100 ml of drinking
water to which the required number of bacteria had been added.
[0083] After infection for 24 hours (D++1), the bottles containing
the contaminated drinking water were replaced with bottles
containing uncontaminated drinking water.
[0084] Seven days after infection (D+7), the spleens of the mice
infected in this way were removed and homogenized separately in 1
ml of physiological saline containing 0.7% of NaCl.
[0085] The number of viable bacteria per spleen was determined by
plating out dilutions of the homogenate, onto TCS agar.
[0086] The effect of the composition of the drinking water on the
splenic colonization by Salmonella was estimated using the
Student's t test.
[0087] For the survival experiments, the mice were infected as
indicated above and the mortality of the animals was recorded for
21 days.
[0088] I-e) Nature of the acids tested
[0089] In addition to the caprylic acid, aromatic acids were
studied for the purpose of comparing their properties to those of
the caprylic acid. The aromatic acids studied were as follows:
benzoic acid, phenylacetic acid, 3-phenylpropionic acid and
4-phenylbutyric acid.
[0090] The aromatic acids and the caprylic acid originate from the
company Sigma. They were all used in the form of their sodium salt
and at a pH of between 7.2 and 7.3.
[0091] II) Results
[0092] II-a) Preliminary step: effect of the concentration of
divalent cations on the expression of the Salmonella invasion
genes
[0093] The purpose of this preliminary step was to demonstrate that
lacZ transcriptional fusions could be used to investigate the
environmental conditions which repress the expression of the genes
present on the SPI-1 pathogenicity island.
[0094] For this, the S. typhi strains Ty267, Ty272 and Ty277, which
carry the iagA'-lacZ, invF'-lacZ and sipB'-lacZ fusions,
respectively, (see Table I), were cultured in the favourable LB or
unfavourable LB medium.
[0095] The .beta.-galactosidase activity expressed by the fusions
was measured at different times of culturing. The results appear in
FIG. 1. Samples were taken every two hours in order to measure the
OD.sub.600 (open symbols on FIG. 1) and in order to determine the
.beta.-galactosidase activity (solid symbols on FIG. 1).
[0096] The S. typhi strains Ty267 (iagA'-lacZ), Ty272 (invF'-lacZ)
and Ty277 (sipB'-lacZ) were cultured in LB medium containing 300 mM
NaCl and 5 mM of CaCl.sub.2 (represented with a square) or 300 mM
NaCl and 1 mM EGTA (represented with a diamond).
[0097] These results show that the expression of the fusions is
very greatly decreased when the culture medium is depleted of
divalent cations.
[0098] They consequently confirm that it is sufficient to modify a
single favourable condition for the invasion genes to no longer be
expressed in S. typhi, as had been demonstrated in S. typhimurium
(Bajaj et al., 1996, mentioned above).
[0099] Consequently, the iagA'-lacZ, invF'-lacZ and sipB'-lacZ
transcriptional fusions in S. typhi were used in the remainder of
this example.
[0100] II-b) Effect of certain aromatic acids and of certain fatty
acids on the expression of the invasion genes
[0101] The S. typhi strain Ty267 (iagA'-lacZ fusion), suspended in
the favourable LB medium, was used to seed the "Biotype 100".RTM.
plates.
[0102] After incubation for 36 hours in an incubator at 37.degree.
C., a clear culture was observed in all the cupules, which
indicates that none of the 99 carbon-based substrates of the plate
has bactericidal or bacteriostatic activity for the Ty267
strain.
[0103] The .beta.-galactosidase activity expressed by the
iagA'-lacZ fusion was determined for the culture in each cupule.
With respect to the .beta.-galactosidase activity measured in the
control cupule without substrate (11 250 .beta.-galactosidase
units), no substrate caused an increase in the expression of the
iagA'-lacZ fusion.
[0104] On the contrary, the expression of this fusion was very
strongly repressed in the cupules containing the sodium
phenylacetate (81 .beta.-galactosidase units), the sodium benzoate
(245 .beta.-galactosidase units), the sodium 3-phenylpropionate
(166 .beta.-galactosidase units) and the sodium caprylate (47
.beta.-galactosidase units).
[0105] Since the amount of substrate contained in each cupule was
not known, a more detailed study of the action of these substrates
on the expression of the invasion genes was undertaken.
[0106] Initially, sodium benzoate and sodium caprylate were
used.
[0107] The effect of the sodium benzoate and that of the sodium
caprylate on the expression of the three fusions is given in FIGS.
2 and 3, respectively.
[0108] On these FIGS. 2 and 3, the OD.sub.600 measurement
corresponds to the open symbols and the .beta.-galactosidase
activity determination corresponds to the solid symbols.
[0109] The S. typhi strains Ty267, Ty272 and Ty277 were seeded in
favourable LB medium and in favourable LB medium containing various
concentrations of sodium benzoate or of sodium caprylate.
[0110] On FIG. 2, the symbols in the form of squares correspond to
the favourable LB medium without the addition of sodium benzoate,
the symbols in the form of diamonds correspond to the favourable LB
medium with the addition of 5 mM of sodium benzoate, the symbols in
the form of circles correspond to the favourable LB medium with the
addition of 2.5 mM of sodium benzoate and the symbols in the form
of triangles correspond to the favourable LB medium with the
addition of 1 mM of sodium benzoate.
[0111] On FIG. 3, the symbols in the form of squares correspond to
the favourable LB medium without the addition of sodium caprylate,
the symbols in the form of diamonds correspond to the favourable LB
medium with the addition of 5 mM of sodium caprylate, the symbols
in the form of circles correspond to the favourable LB medium with
the addition of 2.5 mM of sodium caprylate and the symbols in the
form of triangles correspond to the favourable LB medium with the
addition of 1 mM of sodium caprylate.
[0112] The action of these two compounds on the expression of the
iagA'-lacZ, invF'-lacZ and sipB'-lacZ fusions was studied by
measuring the .beta.-galactosidase activity expressed by these
fusions as a function of the time of culturing (samples removed
every two hours).
[0113] These results show that the presence of one or the other of
these two compounds at the final concentration of 5 mM in the
culture medium did not significantly affect the growth of the S.
typhi strains.
[0114] At a concentration greater than or equal to 2.5 mM, the
sodium benzoate or the sodium caprylate very strongly represses the
expression of the iagA, invf and sipB genes.
[0115] When the favourable LB medium contains 1 mM of sodium
benzoate, the activity of the fusions is again decreased, by 50%
with respect to the values measured in the favourable LB medium
without sodium benzoate.
[0116] If the favourable LB medium contains 1 mM of sodium
caprylate, the .beta.-galactosidase activity expressed by the
fusions remains low, of the order of 20% of that measured in the
favourable LB medium without sodium caprylate.
[0117] If the favourable LB medium is supplemented with 1 mM of
sodium benzoate and with 1 mM of sodium caprylate, the activity of
the iagA'-lacZ fusion measured after culturing for 6 hours is very
low (317 .+-.12 .beta.-galactosidase units).
[0118] This set of results shows that sodium benzoate or sodium
caprylate added at the final concentration of 2.5 mM to the
favourable LB medium very strongly represses the expression of the
Salmonella invasion genes.
[0119] These results also suggest that the inhibitory action of the
sodium caprylate is greater than that of the sodium benzoate, at
least when these products are used at low concentration (1 mM), but
that the effect of these two products is additive.
[0120] II-c) Effects of sodium benzoate and of sodium caprylate on
the specific repression of the expression of the invasion genes of
the SPI-1 pathogenicity island
[0121] In order to examine whether the effect of the sodium
benzoate and of the sodium caprylate was specific for the invasion
genes of the SPI-1 pathogenicity island, the action of these two
products was studied on the tviB'-lacZ transcriptional fusion, in
the S. typhi strain Ty266 (Table I).
[0122] The tviB gene has been characterized in S. typhi, and it
encodes a GDP-dehydrogenase involved in the biosynthesis of the Vi
antigen, which is the capsular polysaccharide of the typhus
bacillus (Waxin et al., Res. Microbiol., 1993, 144, 363-371).
[0123] The results are reported on FIG. 4, in which it is seen that
samples were taken every two hours in order to measure the
OD6.sub.00 (corresponding to the open symbols) and in order to
determine the .beta.-galactosidase activity (corresponding to the
solid symbols). The strains were cultured in favourable LB medium
without the addition of sodium benzoate or of sodium caprylate
(symbols in the form of squares on FIG. 4) and in favourable LB
medium containing 5 mM of sodium benzoate (symbols in the form of
diamonds on FIG. 4) or 5 mM of sodium caprylate (symbols in the
form of circles on FIG. 4).
[0124] As shown in FIG. 4, the sodium benzoate and the sodium
caprylate at 5 mM have no significant effect on the expression of
the tviB'-lacZ fusion.
[0125] Several components of the Inv-Spa-Prg secretion apparatus
show similarities with components of the machinery involved in the
secretion of flagellin and the assembly of flagella.
[0126] In fact, it has been shown in S. typhi that the secretion of
the Sip proteins, the amount of flagellin secreted and the mobility
are regulated in a coordinated way in response to the osmolarity of
the culture medium (Arricau et al., 1998, mentioned above).
[0127] These data led the inventors to study the effect of sodium
benzoate and of sodium caprylate on the mobility of the S. typhi
strain Ty2.
[0128] This study showed that the Ty2 strain had the same mobility
after culturing in a U tube and in a U tube supplemented with 5 mM
of sodium benzoate or with 5 mM of sodium caprylate.
[0129] It results therefrom that sodium benzoate and sodium
caprylate specifically repress the expression of the invasion genes
of the SPI-l pathogenicity island in Salmonella.
[0130] II-d) Effects of certain aromatic acids on the expression of
the invasion genes The S. typhi strain Ty267 (iagA'-lacZ fusion)
was seeded in favourable LB medium and in favourable LB medium
containing the aromatic acid to be tested at the final
concentration of 5 mM.
[0131] The .beta.-galactosidase activity of the iagA'-lacZ fusion
was measured after incubation for 6 hours at 37.degree. C. with
shaking (200 rpm).
[0132] As expected according to the results obtained above in
"Biotype 100".RTM. ( plates, and as appears in Table II
hereinafter, the sodium benzoate, the sodium phenylacetate and the
sodium 3-phenylpropionate inhibit the expression of the iagA'-lacZ
fusion in S. typhi.
[0133] In addition, the .beta.-galactosidase activity of this
fusion is also inhibited by the sodium 4-phenylbutyrate (Table II),
a substrate not appearing in the "Biotype 100".RTM. plate used
above.
2 TABLE II .beta.-galactosidase activity of Aromatic acid tested (5
mM) the iagA'-lacZ fusion None (control favourable LB 12134 .+-.
1811 medium) Sodium benzoate 190 .+-. 17 Sodium phenylacetate 149
.+-. 24 Sodium 3-phenylpropionate 297 .+-. 95 Sodium
4-phenylbutyrate 198 .+-. 32
[0134] These results show that the inhibitory activity of these 4
aromatic acids is very similar.
[0135] The effect of the sodium benzoate, of the sodium
phenylacetate, of the sodium 3-phenylpropionate and of the sodium
4-phenylbutyrate is additive since, if each one of them is added to
the favourable LB medium, at the final concentration of 1 mM, the
activity of the iagA'-lacZ fusion is strongly repressed (236 .+-.10
.beta.-galactosidase units), whereas this fusion is only partially
repressed when the favourable LB medium is supplemented with 1 mM
sodium benzoate alone (5 455 .+-.223 .beta.-galactosidase
units).
[0136] II-e) Compared effects of sodium benzoate and of sodium
caprylate in a model of murine infection with S. typhimurium
C52
[0137] The S. typhimurium strain C52 was used to infect C57Bl/6
mice orally.
[0138] In this experimental model, the 50% lethal dose (LD.sub.50)
of the C52 strain is equal to approximately 4 .times.10.sup.5
bacteria (Coynault et al., Mol. Microbiol., 1996, 22, 149-160) and
the kinetics of colonization of the spleen have been reported
previously (Pardon et al., Ann. Inst. Pasteur/Microbiol., 1986,
137B, 47-60).
[0139] Since sodium benzoate and sodium caprylate repress, in
vitro, the expression of the Salmonella invasion genes, it was
logical to study the effect of these products on the splenic
colonization of C57Bl/6 mice infected orally with S. typhimurium
C52, with 10.sup.8 bacteria (250 LD.sub.50).
[0140] The results are given in FIG. 5. On this figure, the number
of viable bacteria is given as the mean (log.sub.10) of the values
.+-. the standard deviation. Groups of 8 mice were infected orally
with 10.sup.8 viable bacteria (250 LD.sub.50), and the number of
viable bacteria was then determined seven days after infection.
[0141] These results show that the bacterial load in the spleen is
decreased in a highly significant way (p <0.001) in the mice
having received drinking water with a pH of between 7.2 and 7.3
containing a mixture of 50 mm of sodium benzoate and 50 mM of
sodium caprylate, on the condition that the treatment is initiated
two days before infection.
[0142] The administration of sodium benzoate and of sodium
caprylate on the day of or on the day after infection has no
significant effect on the level of splenic colonization.
[0143] The effects of the sodium benzoate and of the sodium
caprylate, administered, in the drinking water, to the mice two
days before infection with S. typhimurium C52, were then studied
separately.
[0144] The effect of EDTA, which, like EGTA, chelates divalent
cations, was studied jointly with the effect of the sodium benzoate
or of the sodium caprylate, during murine infection with S.
typhimurium.
[0145] Groups of five C57Bl/6 mice were infected orally with 5
.times.10.sup.7 bacteria (125 LD.sub.50), according to the protocol
described above. The results obtained are given in Table III
hereinafter:
3 TABLE III Composition of the drinking Number of viable bacteria
water in the spleen (mean log.sub.10) Distilled water (DW) 7.9 .+-.
0.3 DW + 50 mM of sodium 4.5 .+-. 0.9 benzoate + 50 mM of sodium
caprylate DW + 50 mM of sodium 7.8 .+-. 0.8 benzoate DW + 50 mM of
sodium 4.3 .+-. 0.4 caprylate DW + 50 mM of sodium 7.9 .+-. 0.4
benzoate + 10 mM of EDTA DW + 50 mM of sodium 6.7 .+-. 0.9
caprylate + 10 mM of EDTA
[0146] All these compositions have a pH of between 7.2 and 7.3.
[0147] These results show that the administration, two days before
infection and for the duration of the experiment, of drinking water
containing 50 mM of sodium caprylate decreases by a factor of
approximately 1 000 the bacterial load in the spleen on the seventh
day of infection.
[0148] The administration of a mixture of 50 mM of sodium caprylate
and 50 mM of sodium benzoate gives a very comparable result, which
indicates that the sodium benzoate does not act in synergy with the
sodium caprylate to prevent the murine infection with S.
typhimurium C52. In fact, the presence of 50 mM of sodium benzoate
in the drinking water has no effect on the level of splenic
colonization of the C57Bl/6 mice infected with S. typhimurium
C52.
[0149] The latter result should be compared to that which had been
obtained above, in vitro, on the expression of the iagA'-lacZ,
invF'-lacZ and sipB'-lacZ fusions in S. typhi in the presence of
sodium benzoate (FIG. 2).
[0150] As reported in the preliminary experiments (FIG. 1), the
consequence of depleting the favourable medium of divalent cations
with EGTA is the repression of the iagA'-lacZ, invF'-lacZ and
sipB'-lacZ fusions in S. typhi.
[0151] The addition of 10 mM of EDTA to the drinking water
containing 50 mM of sodium benzoate confers no protection against
the infection with S. typhimurium C52 (Table III).
[0152] On the contrary, the presence of 10 mM of EDTA in the
drinking water containing 50 mM of sodium caprylate results in a
decrease in the protective effect observed with the sodium
caprylate alone.
[0153] In the same way, the effect of the concentration of sodium
caprylate in the drinking water was then studied as a function of
the infectious dose.
[0154] The results are given in Table IV hereinafter.
4 TABLE IV Number of viable bacteria in the spleen (mean
log.sub.10) Composition of the Infectious dose Infectious dose
drinking water 5 .times. 10.sup.7 bacteria 10.sup.7 bacteria
Distilled water (DW) 7.7 .+-. 0.3 7.2 .+-. 0.3 DW + 50 mM of sodium
4.6 .+-. 0.3 2.3 .+-. 0.4 caprylate DW + 10 mM of sodium 6.5 .+-.
0.4 6.5 .+-. 1.4 caprylate DW + 5 mM of sodium 6.9 .+-. 0.5 6.8
.+-. 0.2 caprylate
[0155] All these compositions have a pH of between 7.2 and 7.3.
[0156] These results confirm that the administration, two days
before infection and for the duration of the experiment, of
drinking water containing 50 mM of sodium caprylate decreases in a
very highly significant way the bacterial load in the spleen of the
mice infected orally with 5 .times.10.sup.7 bacteria (125
LD.sub.50).
[0157] In addition, these results show that the addition of 50 mM
of sodium caprylate to the drinking water decreases by a factor of
approximately 100 000 the bacterial load in the spleen of the mice
infected orally with 10.sup.7 bacteria (25 LD.sub.50) On the other
hand, the sodium caprylate no longer has a significant protective
effect when the concentration thereof is lowered to 5 or 10 mM in
the drinking water, whatever the infectious dose used.
[0158] II-f) Effect of sodium caprylate on the survival of mice
infected with S. typhimurium The effect of sodium caprylate on the
survival of mice infected with S. typhimurium C52 was then
studied.
[0159] A first batch of ten C57Bl/6 mice, to which drinking water,
with a pH of between 7.2 and 7.3, without sodium caprylate was
administered, was infected orally with 10.sup.7 bacteria (25
LD.sub.50) . No animal survived (3 deaths on the eighth day after
infection, 4 on -the ninth and 3 on the tenth).
[0160] The second batch of 10 mice, to which drinking water with a
pH of between 7.2 and 7.3 containing 50 mM of sodium caprylate had
been administered two days before infection and for the duration of
the experiment, was also infected orally with 10.sup.7 bacteria (25
LD.sub.50) . One mouse died on the twelfth day of infection. On the
twentieth day of infection, the nine remaining mice were sacrificed
and the number of bacteria in the spleen was determined. In the
spleen of one mouse, log.sub.10 =7.3 viable bacteria were counted.
The mean (.+-. standard deviation) of the number of viable bacteria
in the spleen of the other eight animals was calculated to be equal
to log.sub.10 =3.3 .+-.0.3.
[0161] These results show that the presence of 50 mM of sodium
caprylate in the drinking water with a neutral pH makes it possible
to very significantly decrease the bacterial load in the spleen of
C57Bl/6 mice infected orally with S. typhimurium. In addition, the
sodium caprylate effectively protects the animals against infection
with 25 LD.sub.50 of S. typhimurium.
[0162] II-g) Effect of sodium caprylate in a model of murine
infection with S. dublin and S. enteritidis
[0163] The S. dublin strain 5917 (Coynault & Norel, Microb.
Pathog., 1999, 26, 299-305) and S. enteritidis strain LAS (Thorns
et al., Microb. Pathog., 1996, 20, 235-246) were used to infect
C57B1/6 mice orally at the dose of 10.sup.7 bacteria per
animal.
[0164] The results reported are given in Table V hereinafter:
5TABLE V Composition of Number of viable the drinking bacteria in
the Strain water spleen (mean in log.sub.10) S. dublin 5917
Distilled water 7.7 .+-. 0.4 (DW) S. dublin 5917 DW + 50 mM of 2.1
.+-. 0.2 sodium caprylate S. enteritidis DW 7.5 .+-. 0.3 LA5 S.
enteritidis DW + 50 mM of 3.7 .+-. 0.4 LA5 sodium caprylate
[0165] All the compositions used have a pH of between 7.2 and
7.3.
[0166] These results show that the addition of 50 mM of sodium
caprylate to the drinking water at neutral pH very significantly
decreases the bacterial load in the spleen of the mice infected
with S. dublin or with S. enteritidis.
[0167] Consequently, the preventive effect of the sodium caprylate
is independent of the Salmonella serotype, in a murine model of
oral infection.
[0168] III) Conclusion
[0169] This set of results shows that, although sodium benzoate,
sodium phenylacetate, sodium 3-phenylpropionate, sodium
4-phenylbutyrate and sodium caprylate inhibit, in vitro, the
expression of the Salmonella invasion genes, even though, moreover,
the culturing conditions are optimal for the expression of these
genes, none of these compounds has any significant bactericidal or
bacteriostatic effect on the Salmonellae, at neutral pH.
[0170] On the other hand, in a murine model of oral infection,
sodium caprylate (compared to sodium benzoate), added at the
concentration of 50 mM to the drinking water at neutral pH, makes
it possible to considerably decrease the level of splenic
colonization by three major Salmonella serotypes: S. typhimurium,
S. enteritidis and S. dublin.
[0171] It is important to emphasize, in this respect, that the mice
used in these experiments belong to the C57Bl/6 line, which is
particularly sensitive to Salmonella infection (Mastroeni et al.,
Fund. Clin. Immunol., 1994, 2, 83-95).
[0172] In addition, it emerges from these experiments that sodium
caprylate used at neutral pH very effectively protects the animals
against infection with 25 LD.sub.50 of S. typhimurium, since nine
mice out of ten survived this lethal infectious dose.
[0173] On the contrary, sodium benzoate has no protective effect in
the same model of infection.
[0174] Overall, this set of results shows that it is possible to
prevent Gram-negative bacterial infections, such as Salmonella
infections, with sodium caprylate.
EXAMPLE 2: COMPARATIVE STUDY OF THE EFFECT OF CERTAIN FATTY ACIDS
ON THE EXPRESSION OF INVASION GENES
[0175] This study was carried out according to the protocol
described above in Example 1.
[0176] The aim of this study is to demonstrate that only the
compounds corresponding to formula (I) in accordance with the
invention make it possible to inhibit the expression of the
iagA'-lacZ fusion in S. typhi.
[0177] The S. typhi Ty267 strain (iagA'-lacZ fusion) was seeded
into favourable LB medium and into favourable LB medium containing
the fatty acid to be tested, in the form of its sodium salt, at the
final concentration of 5 mM. The various fatty acids tested are
given in Table VI hereinafter:
6TABLE VI Fatty acid substrate Chemical Fatty acid tested for
Salmonella formula Acetic acid (*) Yes CH.sub.3--COOH Propionic
acid (*) Yes CH.sub.3--CH.sub.2--COOH Butyric acid (*) No
CH.sub.3--(CH.sub.2).sub.2--COOH Valeric acid (*) No
CH.sub.3--(CH.sub.2).sub.3--COOH Caproic acid (*) No
CH.sub.3--(CH.sub.2).sub.4--COOH Oenanthic acid (*) No
CH.sub.3--(CH.sub.2).sub.5--COOH Caprylic acid (*) No
CH.sub.3--(CH.sub.2).sub.6--COOH 2-Hydroxycaprylic No
CH.sub.3--(CH.sub.2).sub.5-- acid CH(OH)--COOH 8-Hydroxycaprylic No
CH.sub.2OH--(CH.sub.2).sub.6--COOH acid 2-Aminocaprylic No
CH.sub.3--(CH.sub.2).sub.5-- acid (*) CH(NH.sub.2)--COOH
8-Aminocaprylic No (NH.sub.2)--CH.sub.2--(CH.su- b.2).sub.6-- acid
(*) COOH Pelargonic acid No CH.sub.3--(CH.sub.2).sub.7--COOH (*):
Compound not in accordance with the invention since it does not
correspond to formula (I).
[0178] The .beta.-galactosidase activity of the iagA'-lacZ fusion
was measured after incubation for 6 hours at 37.degree. C. with
shaking (200 rpm).
[0179] The results obtained are given in Table VII hereinafter:
7 TABLE VII .beta.-Galactosidase activity Aromatic acid tested (5
mM) of the iagA'-lacZ fusion None (control favourable LB 22022 .+-.
1177 medium) Acetic acid (*) 29265 .+-. 1692 Propionic acid (*)
14650 .+-. 1333 Butyric acid (*) 21225 .+-. 1741 Valeric acid (*)
509 .+-. 91 Caproic acid (*) 347 .+-. 21 Oenanthic acid (*) 525
.+-. 43 Caprylic acid 445 .+-. 69 2-Hydroxycaprylic acid 225 .+-.
72 8-Hydroxycaprylic acid 593 .+-. 49 2-Aminocaprylic acid (*)
33149 .+-. 2520 8-Aminocaprylic acid (*) 18077 .+-. 1263 Pelargonic
acid 383 .+-. 54
[0180] These results show:
[0181] that the sodium salts of acetic acid, propionic acid and
butyric acid, which are compounds which are not part of the
invention, do not make it possible to inhibit the expression of the
iagA'-lacZ fusion in S. typhi;
[0182] that the sodium salts of valeric acid, caproic acid,
oenanthic acid and pelargonic acid inhibit the expression of the
iagA'-lacZ fusion in S. typhi as strongly as sodium caprylate;
[0183] that the presence of a hydroxyl radical at position 2 or 8
on the caprylic acid molecule (2-hydroxycaprylic acid and
8-hydroxycaprylic acid, respectively) does not modify the
expression of the iagA'-lacZ fusion in S. typhi compared to that
which is obtained with caprylic acid;
[0184] that the presence of an amino radical at position 2 or 8 on
the caprylic acid molecule (2-aminocaprylic acid and
8-amionocaprylic acid, respectively) totally blocks the inhibitory
activity of caprylic acid.
[0185] Consequently, this set of results demonstrates that the
compounds of formula (I) in accordance with the invention make it
possible to suppress, in vitro, the expression of the invasion
genes in a Gram-negative bacterium such as Salmonella.
* * * * *